Molecular recognition is a powerful technique that can be used to generate noncovalently bound host-guest complexes for a variety of purposes. These noncovalent complexes are easily transferred to the gas phase by electrospray ionization (ESI). Unfortunately, attempts to effect intermolecular reactions between the cluster components are often frustrated by the lability of noncovalent complexes, which results from the relatively weak interactions that hold them together.
In the post genomic world of proteomics, many substantial advances will be made through experiments conducted in the gas phase. Therefore, the understanding and control of gas phase peptide chemistry is of paramount importance. For example, the study of gas phase peptide chemistry has revealed that selective cleavage of the peptide backbone will occur at aspartic acid residues (Tsaprailis et al (2000) Int. J. Mass Spectrom. 195/196:467; Tsaprailis et al. (1999) J. Am. Chem. Soc. 121:5142; Lee et al. (1998) J. Am. Chem. Soc. 120:3188). This cleavage occurs by a displacement reaction that yields a stable five-membered ring. Understanding this phenomenon allows for the accurate prediction of peptide cleavages in aspartic acid containing peptides. Furthermore, C-terminal peptide sequencing via a similar mechanism, where the C-terminal amino acids are sequentially removed, has also yielded promising, if limited, results (Lin et al. (1998) Anal. Chem. 70:5162). Unfortunately, this C-terminal sequencing is limited to peptides with eight amino acids or less, severely limiting the utility of this technique for sequencing proteins in the gas phase. The addition of transition metals can also mediate peptide chemistry in the gas phase (Hu et al. (1995) J. Am. Chem. Soc. 117:11314; Nemirovskiy et al. (1998) J. Am. Soc. Mass Spectrom. 9:1285). Preliminary studies have shown that Zn2+, Ni2+, and Co2+ will attach to histidine and promote peptide fragmentation at this residue (Hu et al. (1995) J. Am. Chem. Soc. 117:11314). These experiments were carried out on a very limited sampling of peptides, but the resulting cleavages were highly specific. Similarly, Fe2+ complexes with cysteine containing peptides enhanced the number of cleavages observed at the cysteine residues when the peptide was collisionally activated. These important initial results illustrate that peptide chemistry can be influenced by the addition of appropriate reagents.
A significant amount of work developing reagents that selectively recognize and non-covalently attach to specific amino acid side chains has already been reported (Julian et al. (2001) Int. J. Mass. Spectrom. 210:613-623). Reagents have been developed specificity for the gas phase, as described in Friess et al. (2001) J. Am. Soc. Mass Spectrom. 12(7):810 and Julian et al. (2002) Int. J. Mass Spectrom. 220:87. Solution phase reagents are described in Bell et al. (1999) Angew. Chem. Int. Ed. 38:2543; Galan et al. (1992) J. Am. Chem. Soc. 114:1511; Ludwig et al. (2000) Anal. Chem. 367:103; Ngola et al. (1999) J. Am. Chem. Soc. 121:1192; Rensing et al. (2001) J. Org. Chem. 66:5814; and Schrader (1998) Tetrahedron Lett. 39:517).
The instant invention addresses the needs in the art by the use of reagents that are based upon crown ethers. Crown ethers, and 18-crown-6 ether (18C6) in particular, are well known hosts for protonated primary amines, both in solution and in the gas. More recently, 18C6 was shown to selectively bind to lysine residues in small peptides (Julian et al. (2001) Int. J. Mass Spectrom. 210:613-623).